CN103320468B - UCH320 protein and application of coding gene thereof in adjusting and controlling plant growth and development - Google Patents

UCH320 protein and application of coding gene thereof in adjusting and controlling plant growth and development Download PDF

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CN103320468B
CN103320468B CN201310261912.XA CN201310261912A CN103320468B CN 103320468 B CN103320468 B CN 103320468B CN 201310261912 A CN201310261912 A CN 201310261912A CN 103320468 B CN103320468 B CN 103320468B
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paddy rice
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CN103320468A (en
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王东辉
白书农
刘娜
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Peking University
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    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
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Abstract

The invention discloses UCH320 protein and an application of encoding code thereof in adjusting and controlling plant growth and development. The application provided by the invention is an application of protein consisting of amino acid sequences shown by a sequence 1 in a sequence table or coding genes thereof in adjusting and controlling the plant growth and development. The plant growth and development is specifically embodied in at least one of (1) and (2): (1) the fructification rate of the seed; (2) the weight of the seed. Proven by experiment, in the development process of rice, the expression level of UCH320 protein in the rice is reduced by an RNA (Ribonucleic Acid) interference technology, the rice seed can be caused to show the following phenotype: compared with the wild type rice seed, the fructification rate is reduced, the fertile seed is also long and thin, and the weight of the seed is also obviously reduced. The invention lays foundation for finding out simpler ideas and methods for creating crops with high-yield characters.

Description

The application in regulating growth of plants of UCH320 albumen and encoding gene thereof
Technical field
The invention belongs to molecular biology of plants technical field, relate to a kind of UCH320 albumen and the application of encoding gene in regulating growth of plants thereof.
Background technology
Since the seventies in last century, heterosis utilization is that China's Rice Production has been made great contribution.Face China's Economic development proposes " high yield, high-quality, efficient, safety, ecology " new great demand to agriculture production, can further excavate heterotic application potential with reply, just become the severe challenge of pendulum in face of contemporary science man.
The combination of heterotic formation based on two different parents.To on the basis of existing application, further excavate its potentiality, form machine-processed research except strengthening hybrid vigour, also be badly in need of setting up effective means and create male-sterile character, to effectively expand screening and the outstanding application being combined in production of cross combination.
At present, in breeding work and production, the male-sterile character of widespread use comes from spontaneous mutation and proterties transformation strain thereof more.The source of male-sterile character is very limited, is to expand the particularly serious restrictive factor of application of cross combination screening.According to the rule of the current current international practice, all innovations with application potential are all subject to intellectual property protection.Therefore, find thinking and the method for initiative Artificial Control crop seed size new, that there is independent intellectual property right and output, become and wished to hold one of cannot avoiding of excavating that the countries and regions of hybrid vigour application potential initiative face, key issue urgently to be resolved hurrily.
For a long time, people utilize the method seed selection hybrid crop of conventional breeding always, and these are long in method cycles, it is slow to take effect, can not meet production development in the urgent need to.Gene engineering method is compared traditional method and is had Some features: the seed selection cycle shortens, and fertility is relatively stable, affected by environment little, genotype is relied on few, low in the pollution of the environment.
Summary of the invention
The object of this invention is to provide a kind of UCH320 albumen and the application of encoding gene in regulating growth of plants thereof.
Application provided by the present invention, is specially the protein (called after UCH320 albumen) that is made up of the aminoacid sequence shown in sequence in sequence table 1 or its encoding gene (called after UCH320 gene) at regulating plant following 1)-2) in apply at least one:
1) seed-setting rate;
2) seed weight.
Above-mentioned application is embodied in: the expression amount of the described UCH320 albumen being made up of the aminoacid sequence shown in sequence in sequence table 1 in described plant is lower, and the seed-setting rate of described plant is lower and/or seed weight is lighter; The expression amount of the described UCH320 albumen being made up of the aminoacid sequence shown in sequence in sequence table 1 in described plant is higher, and the drought resistance seed-setting rate of described plant is higher and/or seed weight is heavier.
(the UCH320 albumen) being made up of the aminoacid sequence shown in sequence in sequence table 1 or its encoding gene (UCH320 gene) have following I in seed selection)-II) application in object proterties at least one plant variety also belongs to protection scope of the present invention:
I) seed-setting rate improves or reduces;
II) seed weight increases or reduces.
In actual applications, in the time that the plant variety of institute's seed selection is the plant variety that seed-setting rate improves and/or seed weight increases, the higher plant of described UCH320 expressing quantity need be hybridized as parent.In the time that the plant variety of institute's seed selection is the plant variety that seed-setting rate reduces and/or seed weight reduces, the lower plant of described UCH320 expressing quantity need be hybridized as parent.
A further object of the present invention is to provide a kind of method of cultivating transgenic plant.
The method of cultivation provided by the present invention transgenic plant, specifically can be following (A) or (B):
(A) cultivate and there is following b1)-b2) method of the transgenic plant of at least one in object proterties, comprise the steps:
A), to the encoding gene that imports the protein being formed by the aminoacid sequence shown in sequence in sequence table 1 in object plant, obtain expressing the transgenic plant of described encoding gene;
B) from obtaining, compared with described object plant, thering is following b1 step a) gained transgenic plant)-b2) at least one transgenic plant in object proterties:
B1) seed-setting rate improves;
B2) seed weight increases;
(B) cultivate and there is following d1)-d2) method of the transgenic plant of at least one in object proterties, comprise the steps:
The encoding gene of the protein c) in object plant, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, and obtains transgenic plant;
D) from obtaining, compared with described object plant, thering is following d1 step c) gained transgenic plant)-d2) at least one transgenic plant in object proterties:
D1) seed-setting rate reduces;
D2) seed weight reduces.
In above-mentioned application or method, the encoding gene (UCH320 gene) of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 (UCH320 albumen) is arbitrary described DNA molecular in following (1) to (4):
(1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 102nd to 791 Nucleotide of 5 ' end;
(2) DNA molecular shown in sequence 2 in sequence table;
(3) the protein DNA molecule of the aminoacid sequence composition in the DNA molecule hybridize limiting with (1) or (2) under stringent condition and coding sequence table shown in sequence 1;
(4) there is with the DNA molecular of the arbitrary restriction in (1)-(3) the protein DNA molecule that in 90% above homology and coding sequence table, the aminoacid sequence shown in sequence 1 forms.
Above-mentioned stringent condition can be with 6 × SSC, the solution of 0.5%SDS, and at 65 DEG C, hybridization, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.
Wherein, sequence 2 is made up of 974 Nucleotide, is the cDNA sequence of described UCH320 gene, and wherein 102-791 position is encoding sequence (ORF); Protein shown in sequence 1 in sequence 2 code sequence lists, sequence 1 is made up of 229 amino-acid residues.
In aforesaid method (B), the encoding gene of the described protein in object plant, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, and can be any method that reduces the expression of UCH320 gene described in described object plant.
In the present invention, the encoding gene of the described protein in object plant, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, specifically by realizing in described object plant proceeding to as shown in the formula the DNA fragmentation shown in (I):
SEQ forward-X-SEQ oppositely(I)
Described SEQ forwardthe 14-268 position Nucleotide of sequence 3 in sequence table;
Described SEQ oppositelysequence and described SEQ forwardsequence reverse complemental;
Described X is described SEQ forwardwith described SEQ oppositelybetween intervening sequence, in sequence, described X and described SEQ forwardand described SEQ oppositelyall not complementary.
In the present invention, the nucleotides sequence of the DNA fragmentation shown in described formula (I) is classified the 14-736 position of the sequence 3 in sequence table as.
Sequence 3 is made up of 748 Nucleotide.Wherein, 14-268 position is the forward sequence (SEQ in corresponding above-mentioned formula (1) of a fragment of described UCH320 gene forwardconsistent with the 534-788 position of sequence in sequence table 2), X in the corresponding above-mentioned formula (1) of 269-481 position (276-474 position is GA20 intron nucleotide sequence), 482-736 position is the reverse sequence (SEQ in corresponding above-mentioned formula (1) of a fragment of described UCH320 gene oppositely, be the reverse complementary sequence of the 534-788 position of sequence in sequence table 2).
Further, the DNA fragmentation shown in described formula (I) is to proceed in described object plant by the form of rnai expression carrier; On described rnai expression carrier, starting the promotor that the DNA fragmentation shown in described formula (I) transcribes is Actin promotor.Concrete, described rnai expression carrier is to insert at the multiple clone site place of pCAM23A carrier the recombinant plasmid obtaining after the RNA interference sequence (sequence 3) of described UCH320 gene; More specifically, described rnai expression carrier is to prepare according to the method comprising the steps: with the DNA fragmentation shown in sequence 3 in Spe I and the sequence table of Sal I double digestion, after glue reclaims, be isocaudarner with process Xba I(Xba I with SpeI) be connected with the pCAM23A carrier framework large fragment of Sal I double digestion, obtain described rnai expression carrier.
In aforesaid method (A), the recombinant expression vector of the encoding gene that the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 can be by containing described protein imports in described object plant.
Described recombinant expression vector can be used existing plant expression vector construction.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other plant promoter; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process recombinant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have antibiotic marker thing or the anti-chemical reagent marker gene etc. of resistance.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.
In the method (A) and method (B) of above-mentioned cultivation transgenic plant, to carry the described recombinant expression vector of described UCH320 gene or the described rnai expression carrier of described UCH320 gene imports described object plant, specifically can be: by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity are led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.
In above-mentioned each application or each method, described plant can be monocotyledons, also can be dicotyledons.In the present invention, described plant is specially monocotyledon rice, as spent 11 in rice varieties.
In the present invention, seed-setting rate described in above-mentioned each application or each method be the percentage that real grain number accounts for total number (real grain number+unfilled gtains number) (reference " and Zhang Yi; Shen Fucheng. the relation of Weighing setting percentage and counting setting percentage. hybrid rice; 2006,21 (2): 64-68 ").
Also belong to protection scope of the present invention as shown in the formula the DNA fragmentation shown in (I):
SEQ forward-X-SEQ oppositely(I)
Described SEQ forwardthe 14-268 position Nucleotide of sequence 3 in sequence table;
Described SEQ oppositelysequence and described SEQ forwardsequence reverse complemental;
Described X is described SEQ forwardwith described SEQ oppositelybetween intervening sequence, in sequence, described X and described SEQ forwardand described SEQ oppositelyall not complementary;
The nucleotide sequence of described DNA fragmentation is specially the 14-736 position of the sequence 3 in sequence table.
The recombinant vectors, recombinant bacterium, expression cassette or the transgenic cell line that contain described DNA fragmentation also belong to protection scope of the present invention.
Described recombinant vectors can be both recombinant expression vector, can be also recombinant cloning vector.In one embodiment of the invention, in described recombinant expression vector, starting the promotor that described RNA interference sequence transcribes is Actin promotor, concrete, described recombinant expression vector is to insert at the multiple clone site place of pCAM23A carrier the recombinant plasmid obtaining after the RNA interference sequence (sequence 3) of described UCH320 gene; More specifically, described rnai expression carrier is to prepare according to the method comprising the steps: with the DNA fragmentation shown in sequence 3 in Spe I and the sequence table of Sal I double digestion, after glue reclaims, be isocaudarner with process Xba I(Xba I with Spe I) be connected with the pCAM23A carrier framework large fragment of Sal I double digestion, obtain described rnai expression carrier.
Experimental results show that, in the growth course of paddy rice, by RNA perturbation technique, UCH320 protein expression level in paddy rice is lowered, can cause rice paddy seed to show following phenotype: to compare than wild-type rice paddy seed, setting percentage reduces, even if the seed that can educate is also more tall and thin, the weight of seed also significantly reduces.The present invention finds out thinking and the method for more simply formulating high crop yield proterties to lay a good foundation.
Brief description of the drawings
Fig. 1 is the structure collection of illustrative plates of pUCCRNAi interference carrier.
Fig. 2 is part T 1in generation, proceeds to the PCR qualification result of the transgenic paddy rice of RNAi expression vector pCAM23A-UCH320.Wherein, swimming lane M is DNA molecular amount standard, and each band is followed successively by 5000,3000,2000,1000,750,500,300,200bp from big to small; Swimming lane 1-12 is the positive plant of qualification.
Fig. 3 is the PCR qualification result that part proceeds to the adjoining tree of pCAM23A empty carrier.Wherein, swimming lane M is DNA molecular amount standard, and each band is followed successively by 5000,3000,2000,1000,750,500,300,200bp from big to small; Swimming lane 1-10 is the positive plant of qualification.
Fig. 4 is the each genetic stocks paddy rice of UCH320 gene spike of rice phenotype.Wherein, wt represents to spend No. 11 in not genetically modified wild-type rice varieties; 320R13-1-1 and 320R28-13 are that two embodiment 1 identify positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.
Fig. 5 is the each genetic stocks rice grain of UCH320 gene phenotype.Wherein, wt represents to spend No. 11 in not genetically modified wild-type rice varieties; 320R13-6-1 is that an embodiment 1 identifies positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.
Fig. 6 is that the each genetic stocks rice paddy seed of UCH320 gene weight changes ratio.Wherein, wt represents to spend No. 11 in not genetically modified wild-type rice varieties; 24 " 320R-" is 24 strain embodiment 1 and identifies positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.
Fig. 7 is the each genetic stocks rice paddy seed of UCH320 gene setting percentage.Wherein, wt represents to spend No. 11 in not genetically modified wild-type rice varieties; 24 " 320R-" is 24 strain embodiment 1 and identifies positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
PUCCRNAi interference carrier: obtain from heredity professor Chu Chengcai of institute of the Chinese Academy of Sciences, be documented in " ten thousand is elegant clear etc. for Yan Peiqiang, Bai Xianquan. and application RNAi technology is cultivated anti-TMV virus transgene tobacco. heredity; 2007,29 (8): 1018-1022 " in a literary composition.In pUCCRNAi interference carrier, the recognition site of restriction enzyme Spe I and Bgl II is positioned at intron upstream, and the recognition site of restriction enzyme BamH I and Xba I is positioned at intron downstream.The structure collection of illustrative plates of pUCCRNAi interference carrier as shown in Figure 1.
PCAM23A carrier: Beijing DingGuo ChangSheng Biology Technology Co., Ltd.Be recorded in " Chi Zhengchang. the OsSG01 functional study of paddy rice meiotic gene and analysis. Yangzhou University,, Master's thesis in 2010 " in a literary composition.The promotor that is positioned at Xba I upstream carrying on pCAM23A carrier is Actin promotor.
In rice varieties, spend No. 11: purchased from crop investigations institute of the Chinese Academy of Agricultural Sciences; Within 1979, spent training with No. five/Te Tepu/good fortune of capital wind brocade by the Chinese Academy of Agricultural Sciences crop.Be recorded in " Ni Pichong. Anther Culture new variety-in spend No. 11. Crop Germplasm Resources, 04 phase in 1989 ".
Agrobacterium EHA105: Beijing Quan Shijin biotechnology company limited.
In following embodiment, obtain substratum related in the process of transgenic plant as follows:
1, rice callus induction and subculture medium (japonica rice) NB minimum medium
2, AAM substratum
3, be total to culture medium
4, resistance screening substratum 1L, is poured on disposable flat board about 40-50
5, paddy rice division culture medium (japonica rice) 1L is poured on Boiling tube, approximately 20 left and right
6, paddy rice root media (japonica rice)
1/4MS inorganic salt ?
MS VITAMIN ?
NAA 0.5m?g/L
Paclobutrazol 1m?g/L
Plant gel 2.6g/L
pH 5.8
The related hormone mother liquor compound method of each substratum above:
(1) 0.5mg/ml2,4-D mother liquor join method: take 100mg2,4-D, is placed in small beaker; Adding a small amount of dehydrated alcohol makes it to dissolve completely; 2,4-D spirituous solution is slowly added in the water on magnetic stirring apparatus, if there is precipitation, need to reconfigure; Water is settled to 200ml, 4 DEG C of preservations.
(2) 0.5mg/ml α-NAA mother liquor join method: take 100mgNAA and be placed in small beaker; With the KOH solution dissolving NAA of 1M; Water is settled to 200ml, 4 DEG C of preservations.
(3) 0.5mg/ml6-BA mother liquor join method: take 100mg6-BA and be placed in small beaker; Add a small amount of concentrated hydrochloric acid, grind to form pasty state with glass rod, then add a small amount of concentrated hydrochloric acid, make it to dissolve completely; Dilute with water is also settled to 200ml, 4 DEG C of preservations.
(4) preparation of 100mM Syringylethanone (As): take 196.2mg As, directly dissolve with 5ml DMSO, and be settled to 10ml, be distributed into aseptic tubule ,-20 DEG C of stored frozen.
(5) 5mg/ml KT join method: take 100mg kinetin Kinetin, dissolve with a small amount of 1M KOH, dilute with water is settled to 20ml.After filtration sterilization, be distributed in aseptic tubule-20 DEG C of stored frozen.
The acquisition of embodiment 1, UCH320 transgenic plant and qualification
UCH320 gene source related in the present embodiment is in paddy rice (Oryza.sativa L.), and its cDNA sequence is as shown in sequence in sequence table 2, and sequence 2 is made up of 974 Nucleotide, and wherein 102-791 position is encoding sequence (ORF); Protein (UCH320 albumen) in sequence 2 code sequence lists shown in sequence 1, sequence 1 is made up of 229 amino-acid residues.
One, the structure of RNAi expression vector pCAM23A-UCH320
Design following RNAi primer sequence according to sequence in sequence table 2:
RNAi-23A320-F:5 '-cc aCT AGTaTG GAG GAT GCT CAT TCC-3 ' (underscore place is the recognition sequence of restriction enzyme site Spe I, is thereafter the 534-551 position of sequence 2)
RNAi-23A320-R:5 '-Tc gGA TCCcAC AAC TTT CGA AAG AGC-3 ' (underscore place is the recognition sequence of restriction enzyme site BamH I, is thereafter the reverse complemental of the 771-788 position of sequence 2)
Taking the sequence 2 in sequence table as template, carry out pcr amplification with primed RNA i-23A320-F and RNAi-23A320-R.After cutting PCR product with restriction enzyme Spe I and BamH I enzyme, reclaim object fragment, pUCCRNAi carrier framework large fragment after it is cut with Bgl II enzyme with restriction enzyme Spe I is connected (Bgl II and BamH I are isocaudarners), obtains middle interstitial granules 1.With restriction enzyme BamH I and the Xba I enzyme interstitial granules 1 that hits, reclaim skeleton large fragment again, be connected into the above PCR product of cutting with restriction enzyme Spe I and BamH I enzyme (Xba I and Spe I are isocaudarners), obtain middle interstitial granules 2.Again with restriction enzyme Spe I and the Sal I enzyme interstitial granules 2 that hits, reclaim object fragment (748bp), be connected (Xba I and Spe I are isocaudarners) with the pCAM23A carrier framework large fragment of cutting with Sal I enzyme through restriction enzyme Xba I, obtain recombinant plasmid.The recombinant plasmid called after pCAM23A-UCH320 of the DNA fragmentation shown in sequence 3 in insertion sequence table will be shown through order-checking between the restriction enzyme site Xba I of pCAM23A carrier and Sal I.In RNAi expression vector pCAM23A-UCH320, the promotor that in initiating sequence table, the DNA fragmentation shown in sequence 3 is transcribed is Actin promotor.Wherein, sequence 3 is made up of 748 Nucleotide.Wherein, 14-268 position is the forward sequence (consistent with the 534-788 position of sequence in sequence table 2) of a fragment of described UCH320 gene, 276-474 position is GA20 intron (intron) nucleotide sequence coming from pUCCRNAi carrier, and 482-736 position is the reverse sequence (being the reverse complementary sequence of the 534-788 position of sequence 2 in sequence table) of a fragment of described UCH320 gene.Between forward sequence and reverse sequence, separated to maintain the stability of carrier by one section of intron (intron) sequence; This system produces the dsRNA of band hairpin structure (hairpin) at vegetable cell transcription, cause RNAi, thereby suppresses the expression of goal gene.
Two, the acquisition of transgenic paddy rice and qualification
1, the acquisition of transgenic paddy rice
(1) preparation of rice conversion acceptor
A. the inducing culture of Rice Young Embryo callus
Take away the young fringe threshing of spending No. 11 in the rice varieties of spending about rear 12-15 days, float blighted grain with clear water, with 70% alcohol immersion 1-2 minute, then with being added with 1%(v/v) 1.25% the aqueous sodium hypochlorite solution (active chlorine content is 1.25%(w/v) of Tween20) soak 90 minutes, carry out surface sterilization.(when sterilizing, will often stir) uses aseptic water washing 3-4 time, drains water for subsequent use.On aseptic filter paper, extrude Rice Young Embryo with tweezers and dental scaler and be placed in solid inducing culture (NB minimum medium) above, 26 DEG C of dark evoked callus of cultivating.After about 5-7 days, peel callus, proceed to freshly prepared subculture medium (NB minimum medium) upper, succeeding transfer culture is about 5 days, for common cultivation under the same conditions.
B. the inducing culture of Mature Embryos of Rice callus
The Mature seed of rice shelling is first used 70% alcohol immersion 1-2 minute, then use the aqueous sodium hypochlorite solution (active chlorine content is 30%-40%(w/v) of 30%-40%) soak 30 minutes, carry out surface sterilization (be preferably on shaking table and carry out), aseptic water washing 3-4 time, again seed is placed on aseptic filter paper after suck dry moisture, be placed on mature embryo calli induction media (NB minimum medium) upper, 26 DEG C of dark cultivations (can cultivate by light, light is cultivated and looked fast).After approximately 20 days, peel the callus that mature embryo scultellum grows, proceed to mature embryo subculture medium (NB minimum medium) upper, succeeding transfer culture under the same conditions.Every two weeks succeeding transfer culture once later.Select succeeding transfer culture 4-5 days, the yellowish granular callus of color and luster cultivates altogether.
(2) conversion of Agrobacterium and cultivation
A. extracting and purifying plasmid
Being inoculated in respectively 5ml LB(containing kantlex 50mg/L by containing RNAi expression vector pCAM23A-UCH320 and the bacillus coli DH 5 alpha bacterial classification of pCAM23A empty carrier that step 1 builds) in liquid nutrient medium, 37 DEG C, 200rpm shakes overnight incubation.The plasmid extraction kit of pressing V-GENE company extracts recombinant plasmid.
B. cup soaked in absolute ethyl alcohol is hit in power taking, dries.
C. Agrobacterium EHA105 electric shock preliminary treatment
I. Agrobacterium EHA105 is inoculated in 5ml YEP(containing Streptomycin sulphate Sm50mg/L) in liquid nutrient medium, 28 DEG C, 200rpm concussion overnight incubation to OD600 value is 0.4.
In II.1.5ml centrifuge tube, collect 1ml bacterium liquid, 4 DEG C, 8000rpm, centrifugal 30s.
III. remove raffinate, 200 μ l ddH for precipitation 2o fully suspends, and 4 DEG C, 8000rpm, centrifugal 30s.
IV. repeating step III tri-times.
V. remove raffinate, precipitation ddH 2o fully suspends, and is electric shock Agrobacterium EHA105 competence.Add 200 μ l sterile glycerols to mix to be placed on-80 DEG C for subsequent use.
D. electric shock
I., to 200 μ l EHA105 competence, tip-tap mixes to get plasmid (RNAi expression vector pCAM23A-UCH320 or pCAM23A empty carrier that step 1 builds), is then transferred in electric shock cup, puts on ice.
II. be ready to electric shock device (BioRad), voltage is 2.5V, pins shock button with hand, until a acoustic-electric hits complete.
III. room temperature adds YEP liquid medium after leaving standstill 2min, 28 DEG C of standing 1h, and then 28 DEG C, 200rpm cultivates 2h.
The centrifugal 30s of IV.8000rpm, collects bacterium liquid, precipitation ddH 2o suspends, and is coated with and contains kantlex 50mg/L and the YEB solid medium flat board containing Streptomycin sulphate Sm50mg/L with glass stick, cultivates 48h for 28 DEG C.Scraping lawn Eddy diffusion is in YEB liquid nutrient medium, is cultured to logarithmic growth late period at 28 DEG C; Therefrom get again 0.5ml be forwarded in the same YEB liquid nutrient medium of 100ml after 2-3h to OD600 be 0.5 left and right, by centrifugal 10 minutes of cultured restructuring Agrobacterium 4000g, precipitation suspended into restructuring agrobacterium suspension with the AAM liquid nutrient medium of 100ml.
(3) the common cultivation of Rice Callus and Agrobacterium
Select the good subculture of the state obtaining in step 1 and put into the aseptic triangular flask of 100ml to the Rice Callus of certain hour (succeeding transfer culture 4-5 days, color and luster are yellowish, particulate state), then the restructuring agrobacterium suspension (at least ensuring enough bacterium liquid contacts with material) that adds appropriate step 2 to obtain, places 20min with 80-100r/min room temperature.Take out callus, on aseptic filter paper, suck unnecessary bacterium liquid, transfer to immediately in the common culture medium of solid that is covered with one deck aseptic filter paper, during by callus induction and succeeding transfer culture, being close to all the time the side of substratum still places down, callus should be put neatly, had better not stack each other 25 DEG C of dark culturing 3 days.
(4) screening of resistant calli
Callus after cultivating altogether is fully washed 4-6 time with sterilized water, becomes limpid until wash the aqueous solution of callus, then is aseptic washing 4-5 time of the cephamycin cef of 300mg/L with adding concentration, and 15-20min at every turn blots callus with aseptic filter paper.
Callus is placed in the screening culture medium that contains 25mg/L Totomycin Hygromycin and screens after 14 days and proceed in the screening culture medium that contains 50mg/L Totomycin Hygromycin and continue to screen.2 weeks generation.Most of callus is in latter 10 days brownization of left and right of screening, and milky resistant calli then regrows out at the edge of brownization tissue.Select general lasting 6-8 week.
(5) differentiation of resistant calli
From the resistant calli growing after the screening of two-three-wheel, the resistant calli of selecting milk yellow densification goes to the division culture medium that contains 50mg/L Totomycin Hygromycin and cultivates first dark cultivation 3 days, after forward again 16-20h/d to, intensity of illumination 100-120 μ molm -2s -1illumination condition under cultivate, after 30-40 days, further differentiate seedling.
(6) take root, strong sprout and transplanting
In the time that the bud of resistant calli differentiation grows to about 2-4cm, seedling is moved on on root media, cultivate about two weeks.Select the seedling of high about 10cm, well developed root system, wash away substratum with warm water, in greenhouse, transplant and bury.The water surface, not flood seedling as degree, if become a fine day, need to shade and survive (being as the criterion with guttation) to seedling.
By aforesaid operations, the final two kinds of transgenic seedlings with hygromycin resistance that obtain, proceed to the RNAi expression vector pCAM23A-UCH320 of step 1 structure and the rice plant (T of pCAM23A empty carrier 1generation).
2, the qualification of transgenic paddy rice
The T obtaining from step 1 1in generation, proceeds to the transgenic paddy rice of RNAi expression vector pCAM23A-UCH320, and proceeds in the adjoining tree of pCAM23A empty carrier and extract respectively genomic dna.For the transgenic paddy rice that proceeds to RNAi expression vector pCAM23A-UCH320, carry out pcr amplification with primer 1 and primer 2, obtain through qualification the plant that size is about 460bp object band (with UCH320 forward sequence and GA20 intron sequences) and be the positive plant that proceeds to RNAi expression vector pCAM23A-UCH320.For the adjoining tree that proceeds to pCAM23A empty carrier, use for primer 1 and primer 2 and carry out pcr amplification, show that through qualification (PCR product size the is about 200bp) plant that contains (with GA20 intron sequences) gene is the positive plant that proceeds to pCAM23A empty carrier.
Primer 1:5 '-ACTAGTAGATCTGATGGA-3 ';
Primer 2: 5 '-GGATCCCCTATATAATTTAAG-3 ' (reverse complementary sequence of the 461-481 position of sequence 3).
The T that part steps 1 obtains 1generation proceed to RNAi expression vector pCAM23A-UCH320 transgenic paddy rice qualification result as shown in Figure 2, part proceed to pCAM23A empty carrier adjoining tree qualification result as shown in Figure 3.Through above-mentioned PCR qualification, finally obtain 24 strain PCR and identify positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.
Embodiment 2, transgenic paddy rice Function Identification
Identify positive T with 24 strain embodiment 1 1generation proceeds in the transgenic paddy rice transfer-gen plant of RNAi expression vector pCAM23A-UCH320, not genetically modified wild-type rice varieties spends No. 11, and the adjoining tree that proceeds to pCAM23A empty carrier that embodiment 1 obtains is experiment material.The planting seed of each experiment material is carried out in culture dish to vernalization (every kind of experiment material sowing 80-100 grain), the seedling replanting after vernalization is emerged in flowerpot, then forwards large field, Beijing suburb to and grows.After gathering in the crops the spike of rice of each experiment material plant, it is carried out to the Analysis and Identification of following several respects:
1, spike of rice phenotype analytical
Result shows, and spend compared with No. 11 in not genetically modified wild-type rice varieties, 24 strain embodiment 1 identify positive T 1all there is dysplastic phenotype (Fig. 4) in the spike of rice that generation proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320.And the adjoining tree that proceeds to pCAM23A empty carrier obtaining for embodiment 1, in its spike of rice phenotype and not genetically modified wild-type rice varieties, spend No. 11 basically identical, no difference of science of statistics.
2, seed-setting rate and seed weight statistical study
Result shows, and spend compared with No. 11 in not genetically modified wild-type rice varieties, 24 strain embodiment 1 identify positive T 1generation proceeds on the spike of rice of transgenic rice plant of RNAi expression vector pCAM23A-UCH320 a lot of shrivelled seeds (Fig. 5).Seed on each experiment material plant spike of rice is carried out to setting percentage and hundred grain weight statistical study, and its detailed results is in table 1 and Fig. 6, Fig. 7.Wherein, Figure 6 shows that each experiment material rice paddy seed weight changes ratio, calculates with reference to table 1 result by following formula: rice paddy seed weight changes every hundred real grain weight of ratio=(every hundred real grain weight of every hundred real grain weight-wild-type samples of sample to be tested)/wild-type sample.From above result, can find out, and spend compared with No. 11 in not genetically modified wild-type rice varieties, 24 strain embodiment 1 identify positive T 1seed-setting rate and seed weight that generation proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320 all reduce, even if the seed that can educate also more tall and thin simultaneously.And the adjoining tree that proceeds to pCAM23A empty carrier obtaining for embodiment 1, in its seed-setting rate and seed weight and not genetically modified wild-type rice varieties, spend No. 11 basically identical, no difference of science of statistics.
Seed setting rate and hundred grain weight statistical study on the each experiment material plant of table 1 spike of rice
Plant Real grain number Real grain gross weight g Every hundred real grain weight g Unfilled gtains number Total grain number Setting percentage
320R13-1-1 516 11.518 2.232171 1126 1642 31.43%
320R13-1-2 534 11.354 2.126217 1212 1746 30.58%
320R13-1-3 1015 22.041 2.171527 2165 3180 31.92%
320R13-3-1 607 13.828 2.278089 1295 1902 31.91%
320R13-3-2 714 16.352 2.290196 1400 2114 33.77%
320R13-3-3 686 16.132 2.351603 1104 1790 38.32%
320R13-3-4 614 13.689 2.229479 1123 1737 35.35%
320R13-3-5 760 17.748 2.335263 1186 1946 39.05%
320R13-4-1 473 10.38 2.194503 988 1461 32.38%
320R13-4-2 172 3.47 2.017442 646 818 21.03%
320R13-6-1 144 3.31 2.298611 642 786 18.32%
320R13-6-2 190 4.19 2.205263 588 778 24.42%
320R27-A 31 0.66 2.129032 678 709 4.37%
320R28-13 395 9.822 2.486582 2530 2925 13.50%
320R28-B 64 1.69 2.640625 1122 1186 5.40%
320R29-1-1 70 1.653 2.361429 1458 1528 4.58%
320R29-1-2 93 2.16 2.322581 630 723 12.86%
320R29-1-3 106 2.643 2.493396 2190 2296 4.62%
320R29-4-1 21 0.457 2.17619 2365 2386 0.88%
320R29-4-2 107 2.83 2.64486 1235 1342 7.97%
320R29-4-3 40 0.896 2.24 1450 1490 2.68%
320R29-D 1435 36.879 2.569965 1428 2863 50.12%
320R29-F-1 90 2.12 2.355556 1610 1700 5.29%
320R29-F-2 826 20.924 2.533172 961 1787 46.22%
wt 2543 70.4411 2.77 23 2566 99.10%
Note: 1,24 " 320R-" in table are 24 strain embodiment 1 and identify positive T 1in generation, proceeds to the transgenic rice plant of RNAi expression vector pCAM23A-UCH320; Wt represents to spend No. 11 in not genetically modified wild-type rice varieties.2, " total grain number " in table equals " real grain number (having a meter seed) " and adds " unfilled gtains number (without a rice ghost) "; " setting percentage " equals " real grain number " divided by " total grain number ".

Claims (8)

1. the protein being formed by the aminoacid sequence shown in sequence in sequence table 1 or the application of its encoding gene in adjusting and controlling rice grows;
Described rice growth, is embodied in following 1)-2) at least one:
1) seed-setting rate;
2) seed weight.
2. the protein or its encoding gene that are made up of the aminoacid sequence shown in sequence in sequence table 1 have following I in seed selection)-II) application in object proterties at least one rice varieties:
I) seed-setting rate improves or reduces;
II) seed weight increases or reduces.
3. application according to claim 1 and 2, is characterized in that: the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 is following (1) or (2) described DNA molecular:
(1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 102nd to 791 Nucleotide of 5 ' end;
(2) DNA molecular shown in sequence 2 in sequence table.
4. cultivate the method for transgenic paddy rice, for following (A) or (B):
(A) cultivate and there is following b1)-b2) method of the transgenic paddy rice of at least one in object proterties, comprise the steps:
A), to the encoding gene that imports the protein being formed by the aminoacid sequence shown in sequence in sequence table 1 in object paddy rice, obtain expressing the transgenic paddy rice of described encoding gene;
B) a) gained transgenic paddy rice, obtain, compared with described object paddy rice, thering is following b1 from step)-b2) at least one transgenic paddy rice in object proterties:
B1) seed-setting rate improves;
B2) seed weight increases;
(B) cultivate and there is following d1)-d2) method of the transgenic paddy rice of at least one in object proterties, comprise the steps:
The encoding gene of the protein c) in object paddy rice, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, and obtains transgenic paddy rice;
D) c) gained transgenic paddy rice, obtain, compared with described object paddy rice, thering is following d1 from step)-d2) at least one transgenic paddy rice in object proterties:
D1) seed-setting rate reduces;
D2) seed weight reduces.
5. method according to claim 4, is characterized in that: the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 is following (1) or (2) described DNA molecular:
(1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 102nd to 791 Nucleotide of 5 ' end;
(2) DNA molecular shown in sequence 2 in sequence table.
6. method according to claim 4, it is characterized in that: the encoding gene of the described protein in object paddy rice, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, and is by proceeding in described object paddy rice and realize as shown in the formula the DNA fragmentation shown in (I):
SEQ forward-X-SEQ oppositely(I)
Described SEQ forwardthe 14-268 position Nucleotide of sequence 3 in sequence table;
Described SEQ oppositelysequence and described SEQ forwardsequence reverse complemental;
Described X is described SEQ forwardwith described SEQ oppositelybetween intervening sequence, in sequence, described X and described SEQ forwardand described SEQ oppositelyall not complementary.
7. method according to claim 6, is characterized in that: the nucleotides sequence of the DNA fragmentation shown in described formula (I) is classified the 14-736 position of the sequence 3 in sequence table as.
8. method according to claim 6, is characterized in that: the DNA fragmentation shown in described formula (I) is to proceed in described object paddy rice by the form of rnai expression carrier; On described rnai expression carrier, starting the promotor that the DNA fragmentation shown in described formula (I) transcribes is Actin promotor.
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Accession Number NM_00105414;Tanaka,T.等;《Genbank》;20100608;3页 *
Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana;The Rice Annotation Project;《Genome Research》;20070108;第17卷;175-183页 *
Tanaka,T.等.Accession Number NM_00105414.《Genbank》.2010,1-3页. *
The Rice Annotation Project.Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana.《Genome Research》.2007,第17卷175-183页. *

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